Chimeric antigen receptor, nucleic acid molecule, CAR-Ms and their applications

Abstract

The present invention provides a chimeric antigen receptor, a nucleic acid molecule, CAR-Ms, and applications thereof, and belongs to the field of biomedical engineering and peptides. The chimeric antigen receptor provided by the present invention comprises a CD8 α leader signal peptide, a single-chain variable fragment, a CD8 α hinge, a CD8 transmembrane domain, and an intracellular co-stimulatory signal transduction domain. The single-chain variable fragment can target any known antigen; the intracellular co-stimulatory signal transduction domain is any one or a combination of TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, and CD3ζ. The present invention also provides a macrophage-targeted nanogel for RNA delivery. Macrophages transfected with the nanogel system express chimeric antigen receptors, which can achieve specific targeting and phagocytosis of known antigen-positive (e.g., MUC1) tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering technology and peptides, and relates to a chimeric antigen receptor, a nucleic acid molecule, CAR-Ms (chimeric antigen receptor-macrophage) and their applications. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Malignant tumors are a major global health challenge, with their incidence and mortality rates continuing to rise. According to the latest data, common cancers such as breast cancer pose a serious threat to women's health, while lung cancer is the leading cause of cancer death. Traditional treatments such as surgery, radiotherapy, and chemotherapy remain the mainstays, but emerging approaches such as targeted therapy and immunotherapy are gaining traction. CAR-T therapy (chimeric antigen receptor T-cell immunotherapy) has demonstrated significant efficacy in treating leukemia and multiple myeloma, and several CAR-T therapies have been approved for marketing worldwide. However, immunotherapy still faces challenges. Some tumors may develop resistance to CAR-T therapy, resulting in reduced treatment efficacy. Furthermore, CAR-T therapy can cause serious side effects such as cytokine release syndrome.

[0004] Macrophages are core members of the immune system, phagocytizing pathogens (such as bacteria and viruses), clearing senescent cells, presenting antigens, and regulating immunity. Their plasticity allows them to differentiate into either a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype depending on microenvironmental signals, making them particularly crucial in tumor immunity. Conventional CAR-T cells have limited effectiveness against solid tumors due to their dense stroma and immunosuppressive microenvironment, characterized by infiltration of MDSCs (myeloid-derived suppressor cells) and Tregs (regulatory T cells), which hinder immune cell infiltration. Macrophages, on the other hand, naturally have the ability to migrate into tumors (macrophages can comprise up to 40%-50% of a tumor), making them ideal vehicles for penetrating solid tumor barriers. Using gene-editing techniques (such as adenoviral vectors and lipid nanoparticles), chimeric antigen receptors (CARs) can be introduced into macrophages, conferring tumor-targeting capabilities and specific recognition of tumor-associated antigens. They can also induce M1 polarization through costimulatory signals, enhancing phagocytic activity and secretion of proinflammatory cytokines. In addition, CAR-Ms can present tumor antigens to T cells, activating adaptive immune responses, achieving a "killing three birds with one stone" effect. However, the structural design of CAR-Ms is a complex system with multiple modules that require a balance between targeting, activation signals, safety, and scalability. The industry is still in urgent need of developing CAR-Ms with superior anti-tumor effects. Summary of the Invention

[0005] To address the above problems, the present invention provides a chimeric antigen receptor, a nucleic acid molecule, CAR-Ms, and their applications. CAR-Ms obtained using the nucleic acid molecule and its nanogel formulation provided by the present invention can express CAR proteins targeting known antigens and have the ability to recognize and phagocytose tumor cells with known antigens.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a chimeric antigen receptor comprising: a CD8α leader signal peptide, a single-chain variable fragment, a CD8α hinge region, a CD8 transmembrane region, and an intracellular co-stimulatory signaling domain;

[0008] Wherein, the single-chain variable fragment can target any known antigen;

[0009] The intracellular costimulatory signaling domain is selected from at least one of TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, and CD3ζ;

[0010] The amino acid sequence of the CD8α leader signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO: 2, the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO: 3, and the amino acid sequences of TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, and CD3ζ are shown in SEQ ID NO: 4 to SEQ ID NO: 13.

[0011] Preferably, the single-chain variable fragment is derived from a single-chain antibody of MUC1, comprising a light chain variable region and a heavy chain variable region, connected by a linker sequence;

[0012] The intracellular co-stimulatory signaling domain is selected from the group consisting of a combination of Mincle and CD40, a combination of Mincle and 4-1BB, a combination of Mincle and OX40, a combination of Dectin-1 and CD40, a combination of Dectin-1 and 4-1BB, a combination of Dectin-1 and OX40, a combination of TLR2 and CD40, a combination of TLR2 and 4-1BB, a combination of TLR2 and OX40, a combination of TLR4 and CD40, a combination of TLR4 and 4-1BB, a combination of TLR4 and OX40, a combination of TLR7 and CD40, a combination of TLR7 and 4-1BB, a combination of TLR7 and OX40, a combination of NOD2 and CD40, a combination of NOD2 and 4-1BB, and a combination of NOD2 and OX40.

[0013] The second aspect of the present invention provides a nucleic acid molecule comprising: a gene encoding the chimeric antigen receptor described above;

[0014] The CD8α leader signal peptide nucleotide is shown in SEQ ID NO: 14, the CD8α hinge region nucleotide sequence is shown in SEQ ID NO: 15, the CD8 transmembrane region nucleotide sequence is shown in SEQ ID NO: 16, and the TLR2, TLR4, TLR7, Mincle, Dectin-1, NOD2, CD40, 4-1BB, OX40, and CD3ζ nucleotide sequences are shown in SEQ ID NO: 17-SEQ ID NO: 26.

[0015] The third aspect of the present invention provides a CAR-Ms, wherein the CAR-Ms expresses the chimeric antigen receptor described above;

[0016] Alternatively, the CAR-Ms contains the above-mentioned nucleic acid molecule.

[0017] Preferably, the CAR-Ms is constructed using the following method:

[0018] The natural calreticulin, the cross-linking agent, and the above-mentioned nucleic acid molecules are uniformly mixed in a solvent, reacted, and then surface-modified with 4-(2-aminoethyl)benzenesulfonamide to obtain a nanogel preparation;

[0019] The nanogel preparation is used to transfect macrophages to obtain CAR-Ms.

[0020] The fourth aspect of the present invention provides the use of the above-mentioned nucleic acid molecules in the preparation of nanogel preparations, comprising:

[0021] The natural calreticulin, the cross-linking agent and the above nucleic acid molecules are uniformly mixed in a solvent, reacted, and then surface modified with 4-(2-aminoethyl)benzenesulfonamide to obtain a nanogel preparation.

[0022] The fifth aspect of the present invention provides the use of the above-mentioned chimeric antigen receptor in the preparation of an anti-tumor pharmaceutical composition.

[0023] Preferably, the tumor is at least one selected from lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, nervous system cancer, leukemia, and cervical cancer.

[0024] Preferably, the anti-tumor pharmaceutical composition comprises: immune cells, wherein the immune cells express the above-mentioned chimeric antigen receptor.

[0025] More preferably, the immune cells are selected from at least one of T cells, γδ T cells, macrophages, NK cells, NKT cells, regulatory T cells, and neutrophils.

[0026] Beneficial effects of the present invention

[0027] (1) The CAR-Ms obtained from the nucleic acid molecules and nanogel preparations provided by the present invention can express CAR proteins targeting known antigens and have the ability to recognize and phagocytose tumor cells with known antigens.

[0028] (2) The present invention is highly practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 Schematic diagram of the CAR gene.

[0031] Figure 2 Transmission electron microscopy image of the nanogel preparation.

[0032] Figure 3 is the expression rate of CAR protein in CAR-Ms.

[0033] Figure 4 The results of CAR-Ms phagocytosis of tumor cells.

[0034] Figure 5 The effect of CAR-Ms in treating orthotopic pancreatic cancer in mice. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] In a first aspect, the present invention provides a chimeric antigen receptor, the partial amino acid sequences of which are shown in SEQ ID NO:1-SEQ ID NO:13. The scFv (single-chain variable fragment) used in this invention can be derived from various monoclonal antibodies known in the art, including but not limited to monoclonal antibodies targeting HER2, MUC1, GPC3, EGFRvIII, ROR1, CD171, B7-H3, integrin αvβ3, GPC1, Claudin18.2, CD19, BCMA, and the like. In one embodiment validated by the present invention, the scFv is derived from the tumor-specific MUC1 monoclonal antibody 5E5.

[0037] According to general understanding in the art, the MUC1-CAR protein includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a single-chain variable fragment (scFv) of a monoclonal antibody and a hinge region that acts as a linker. In a preferred embodiment of the present invention, the single-chain variable fragment is a single-chain antibody of MUC1, comprising a light chain variable region (5E5 VL) and a heavy chain variable region (5E5 VH), connected by a linker sequence. The hinge region is selected from CD8α.

[0038] Preferably, the transmembrane domain is derived from one of CD4, CD8, CD28 or CD3ζ; further, the transmembrane structure is selected from CD8.

[0039] Preferably, the intracellular domain is a signal transduction domain, and further, the signal transduction domain is the CD3ζ intracellular region.

[0040] Preferably, the co-stimulatory domains are TLR7 and CD40.

[0041] In one embodiment of the above preferred technical solution, the CAR protein is a fusion protein of CD8α signal peptide, anti-MUC1 single-chain antibody, myc-tag marker gene, CD8α hinge region, CD8 transmembrane region, CD3ζ intracellular region, TLR7 and CD40 co-stimulatory region, P2A and EGFP connected in sequence.

[0042] In the above embodiment, the above-mentioned parts of the fusion protein, such as the CD8α signal peptide, the light chain variable region and the heavy chain variable region of the single-chain antibody, the myc-tag marker gene, the CD8α hinge region, the CD8 transmembrane region, the CD3ζ intracellular region, the co-stimulatory region, P2A and EGFP, etc., can be directly connected to each other, or can be connected through a linker sequence. The linker sequence can be a linker sequence suitable for antibodies well known in the art, such as a linker sequence containing G and S. Typically, the linker contains one or more repeated motifs. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motif is adjacent in the linker sequence, and no amino acid residues are inserted between the repeats. The linker sequence can be composed of 1, 2, 3, 4 or 5 repeating motifs. The length of the linker can be 3-25 amino acid residues, for example 3-15, 5-15, 10-20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycine residues in the linker sequence is not particularly limited and is generally 2-20, for example, 2-15, 2-10, or 2-8. In addition to glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q). In certain embodiments, the light chain variable region and the heavy chain variable region of the anti-MUC1 single-chain antibody of the present invention are linked by (GGGGS)n, where n is an integer from 1 to 5.

[0043] It should also be understood that in gene cloning operations, it is often necessary to design appropriate restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, but will not affect the activity of the target sequence. In addition, in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, such as suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Based on the above design, the amino or carboxyl termini of the fusion proteins of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in the present invention. The tags can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, and Ty1. These tags can be used to purify proteins.

[0044] The present invention also includes mutants of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 13. The mutants include: an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence similarity to the CAR and retaining the biological activity of the chimeric antigen receptor. The similarity of the mutant sequences can be calculated using BLAST, such as that of NCBI.

[0045] The above-mentioned mutants also include: an amino acid sequence having one or more mutations (insertions, deletions or substitutions) in the amino acid sequence shown in SEQ ID NO: 1-SEQ ID NO: 13, while still retaining the biological activity of the CAR; the several mutations generally refer to within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, in the art, conservative substitutions are performed with amino acids with similar or similar properties. When substituted, the function of the protein or polypeptide is generally not changed. "Amino acids with similar or similar properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in a polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

[0046] A second aspect of the present invention provides a nucleic acid molecule comprising the nucleotide sequences shown in SEQ ID NO: 14 to SEQ ID NO: 16 and any one of the nucleotide sequences shown in SEQ ID NO: 17 to SEQ ID NO: 26 or a combination thereof.

[0047] In some embodiments, the nucleic acid molecules of the nucleotide sequences shown in SEQ ID NO: 14 to SEQ ID NO: 16 and any one of the nucleotide sequences shown in SEQ ID NO: 17 to SEQ ID NO: 26, or a combination thereof, include DNA and RNA. Further, the DNA and RNA include plasmid DNA, mRNA, circular RNA, etc.

[0048] In some embodiments, the RNA comprises at least one chemical modification selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0049] In some aspects, the mRNA comprises at least one chemically modified nucleoside, wherein the at least one chemically modified nucleoside is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some aspects, the at least one chemically modified nucleoside is N1-methylpseudouridine. In some aspects, the polynucleotide is a fully modified N1-methylpseudouridine mRNA.

[0050] In some embodiments, the nucleic acid molecule may comprise the nucleic acid sequence of one or more of the following marker genes: EGFP, NLS-EGFP, sfGFP, sfGFP, TurboGFP, hrGFP, d2EGFP, ZsGreen1, EGFP(S65T), mNeonGreen, Venus, EYFP, YPet, Cerulean, CyPet, EBFP, TagBFP, TagBFP2, NLS_TagBFP2, dTomato, tdTomato, NLS_tdTomato, DsRed_Express2, TurboRF P, mRFP1, mCherry, NLS_mCherry, mApple, mKate2, Neo, Puro, Hygro, Bsd, Bar, Neo / Kana, Hygro, FKBP / Casp8, FKBP / Casp8, deltaTK, CodA, DTA, DTR, Luciferase, Luc2, MetLuc, Rluc, Nluc, Aequorin, hRluc, LacZ, SEAP, GUSPlus, pHluorin2, and Superecliptic-pHluorin reporter genes.

[0051] Preferably, EGFP or Luciferase is used as a reporter gene.

[0052] The third aspect of the present invention provides a CAR-Ms, wherein the CAR-Ms expresses the chimeric antigen receptor as described in the first aspect.

[0053] A fourth aspect of the present invention provides a gel material. The lipid material is prepared from natural calreticulin by a cross-linking agent and is surface-modified using 4-(2-aminoethyl)benzenesulfonamide.

[0054] In some embodiments, the native calreticulin can be from different species, including human, mouse, rat, chicken, cow, mammal, rabbit, etc.

[0055] The fifth aspect of the present invention provides a nanogel preparation, wherein each nanoparticle comprises the nucleic acid molecule described in the second aspect and the gel material described in the fourth aspect. In some embodiments, each nanoparticle comprises one or more excipients.

[0056] The sixth aspect of the present invention provides a method for constructing CAR-Ms, comprising the following steps: using the nucleic acid molecule provided by the second aspect of the present invention, or the gel material provided by the fourth aspect, or the nanogel preparation provided by the fifth aspect to transfect macrophages to obtain CAR-Ms.

[0057] Methods for introducing genes into cells and expressing them in cells are known in the art. Vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. In some embodiments, the nucleic acid molecules provided in the second aspect of the present invention can be delivered using viral vectors (including but not limited to lentiviruses, adenoviruses, and retroviruses), lipid nanoparticles, cationic polymers (including but not limited to chitosan, hyaluronic acid, hydroxybenzoic acid, dextran, cyclodextrin, polyethyleneimine, polylysine, polyamide, and poly-β-amino esters), inorganic polymers (including but not limited to calcium phosphate, mesoporous silica, gold nanoparticles, and quantum dots), and exosomes.

[0058] Preferably, the nanogel preparation provided by the fifth aspect of the present invention is used to transfect macrophages.

[0059] In a seventh aspect, the present invention provides a CAR-Ms or a drug combination containing the CAR-Ms.

[0060] The CAR-Ms of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as related cytokines or cell populations. Simply put, the pharmaceutical composition of the present invention includes the above-mentioned CAR-Ms, with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Since the chimeric antigen receptor-modified macrophages described in the present invention are used as active substances, in order to maintain the activity of such macrophages, the composition may also include a buffer (such as neutral buffered saline, sulfate buffered saline, etc.), a carbohydrate (such as glucose, mannose, sucrose or dextran, mannitol), a protein, a polypeptide or an amino acid (such as glycine), an antioxidant, a chelating agent (such as EDTA or glutathione), an adjuvant (e.g., aluminum hydroxide) or a preservative. One or more.

[0061] The eighth aspect of the present invention provides a method of administration according to the seventh aspect of the present invention, including by spraying, injection, swallowing, infusion, implantation or transplantation; further, the composition can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. The dosage of the active ingredient in the pharmaceutical composition can be determined by conventional research methods in the art. The amount and frequency of administration of the pharmaceutical composition will be determined by factors such as the patient's condition, and the type and severity of the patient's disease.

[0062] Preferably, the seventh aspect of the present invention is administered by intravenous injection.

[0063] The ninth aspect of the present invention provides the chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the CAR-Ms described in the third aspect, the nanogel preparation described in the fifth aspect, the construction method of the CAR-Ms described in the sixth aspect, and the use of the CAR-Ms or a drug combination containing the CAR-Ms described in the seventh aspect in the preparation of anti-tumor products, wherein the anti-tumor products include but are not limited to drugs or model drugs.

[0064] The tenth aspect of the present invention provides a method for treating tumors with immune cells, which comprises administering the nanogel preparation of the fifth aspect to a patient in need of treatment, or reinfusing the CAR-Ms of the seventh aspect or a drug combination containing the CAR-Ms.

[0065] Preferably, the tumor includes but is not limited to one of lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, nervous system cancer, leukemia, and cervical cancer.

[0066] Preferably, the treatment method further comprises obtaining macrophages of the patient, and transferring the nucleic acid molecule of the second aspect or the nanogel preparation of the fifth aspect into the macrophages.

[0067] The present invention also includes a type of cell therapy in which immune cells are genetically modified in vivo to express the fusion protein described in the first aspect of the present invention. In some embodiments, the immune cells can be T cells, γδ T cells, macrophages, NK cells, NKT cells, regulatory T cells, neutrophils, etc.

[0068] Preferably, the immune cells are macrophages.

[0069] In some embodiments, the CAR-Ms or pharmaceutical compositions containing the CAR-Ms described herein can be combined with other therapies known in the art, including but not limited to chemotherapy, radiotherapy, and immunosuppressants.

[0070] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0071] Example 1: CAR structure design

[0072] (1) CAR protein structure Figure 1 The figure shows the CD8α signal peptide, scFv fragment, CD8α hinge region, CD8 transmembrane region, and intracellular costimulatory signaling domain (costimulatory region and CD3ζ intracellular region) connected in sequence. The scFv fragment is derived from the MUC1 monoclonal antibody 5E5 and includes a light chain variable region (5E5 VL) and a heavy chain variable region (5E5 VH), connected by (GGGGS)3. The costimulatory region in the intracellular costimulatory signaling domain is designed as shown in Table 1.

[0073] Table 1. Design and numbering of co-stimulatory regions in the intracellular structure of CAR protein

[0074]

[0075] Construction of CAR gene mRNA

[0076] A CAR gene expression vector was constructed. In this example, the scFv segment was derived from the 5E5 antibody targeting MUC1, and EGFP was used as a reporter gene. An enzyme digestion system was added, and the mixture was centrifuged overnight. The linearized plasmid was purified and recovered using a QIAquick column, and the purity of the linearized plasmid was subsequently verified by agarose gel electrophoresis. The linearized plasmid was transcribed in vitro using an mRNA transcription system, and the mRNA purity was again verified by agarose gel electrophoresis. A capping system was configured to cap the 5' end of the mRNA, and the capped mRNA was purified using an RNeasy Mini spin column. The mRNA was collected and purity was verified again for subsequent use.

[0077] Example 2. Preparation of Nanogel Preparation

[0078] N-hydroxysuccinimide-disulfide-N-hydroxysuccinimide (NHS-SS-NHS, 93.5 μg) dissolved in 9.35 μL dimethyl sulfoxide (DMSO) was added to phosphate buffered saline (PBS, pH 7.4, 132 μL) containing calreticulin (1,320 μg), and mRNA was added. The mixture was rotated and incubated at 25°C for 30 minutes, and then diluted with 1,188 μL PBS (phosphate) buffer. The resulting nanogel product was buffer exchanged through an ultrafiltration tube: repeated centrifugation and washing with 1.5 mL PBS three times to remove unreacted small molecules to obtain nanogel (NG). Subsequently, 4-(2-aminoethyl)benzenesulfonamide (ABS) was introduced by Michael addition reaction, NG and ABS were dissolved in methanol and reacted in the dark for 24 hours, impurities were removed using a dialysis membrane (MWCO = 500), and then freeze-dried to obtain nanogel (ANG). The morphology of the nanogel is as follows Figure 2 As shown, the average particle size is about 120nm.

[0079] Example 3. Detection of transfection efficiency of nanogel preparation

[0080] Human peripheral blood macrophages (HPBDMs) were seeded into 6-well plates (1×10 6 The DMEM medium containing the nanogels was filtered through a 0.22 μm microporous filter membrane and then added to a 6-well plate containing HPBDMs (1 mL per well). The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 4 h. The cells were then washed 3 times with PBS to remove the unphagocytosed nanogels and replaced with fresh DMEM medium - 10% FBS (fetal bovine serum), 10 ng / ml M-CSF (macrophage colony-stimulating factor). The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for another 24 h. PBS was used as the control group, and the samples corresponding to CAR proteins No. 1-18 in Table 1 were used as the treatment group. Flow cytometry was used to detect the transfection efficiency of each group of nanogel preparations using EGFP fluorescence signals. The transfection results are shown in Figure 2. Figure 3 As shown, the nanogel formulation can achieve efficient transfection of macrophages.

[0081] Example 4. Detection of specific phagocytosis of tumor cells by CAR-Ms

[0082] MUC1 + PANC-1 cells were pre-labeled with Calcein AM (2 μM) and then seeded in 12-well plates (5 × 10 cells per well). 4 cells), different groups of CAR-Ms were added to tumor cells (5×10 4The cells were incubated for 4 h in a cell culture incubator at 37°C and 5% CO2. The cells were then harvested, labeled with Anti-hCD68-PerCP-Cy5.5, and analyzed for phagocytosis using flow cytometry. PBS served as the control group, and samples corresponding to CAR proteins 1-18 in Table 1 served as the treatment groups. The experimental results are shown in Table 1. Figure 4 As shown, macrophages transfected with the nanogel formulation showed a strong affinity for MUC1. + Specific recognition and phagocytosis of tumor cells, among which CAR-Ms No. 5 showed the strongest phagocytic effect.

[0083] Example 5. Detection of the anti-tumor effect of nanogels

[0084] CAR-Ms were injected into mice bearing orthotopic pancreatic cancer tumors 7 days after tumor implantation, and the drug was administered once every 7 days for a total of 2 times. The normal saline group was used as the control group, and the samples corresponding to CAR proteins No. 1-18 in Table 1 were used as the treatment group. On the 15th day after tumor implantation, the mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (0.5 mL / mouse), followed by intraperitoneal injection of 0.1 mL of D-luciferin potassium salt in normal saline solution (30 mg / mL). The fluorescence signal intensity of the tumor tissue was recorded by the IVIS detection system to evaluate the tumor growth. The experimental results are shown in Figure 2. Figure 5 As shown, CAR-Ms transfected with the nanogel formulation can effectively inhibit tumor growth, among which CAR-Ms No. 5 showed the strongest tumor inhibitory effect.

[0085] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A CAR-Ms, characterized in that: The CAR-Ms express a chimeric antigen receptor; The CAR-Ms are constructed using the following method: The natural calreticulin, a cross-linking agent, and a nucleic acid molecule are uniformly mixed in a solvent, reacted, and then surface modified with 4-(2-aminoethyl)benzenesulfonamide to obtain a nanogel preparation; The nanogel preparation is used to transfect macrophages to obtain CAR-Ms; The chimeric antigen receptor comprises: a CD8α leader signal peptide, a single-chain variable fragment, a CD8α hinge region, a CD8 transmembrane region and an intracellular co-stimulatory signal transduction domain; Wherein, the single-chain variable fragment can target any known antigen; The signal transduction domain is the intracellular region of CD3ζ; the costimulatory domain is a combination of TLR7 and CD40; The amino acid sequence of the CD8α leader signal peptide is shown in SEQ ID NO: 1, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO: 2, the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO: 3, and the amino acid sequences of TLR7, CD40, and CD3ζ are shown in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 13; The nucleic acid molecule includes a gene encoding a chimeric antigen receptor expressed by the CAR-Ms; The CD8α leader signal peptide nucleotide is shown in SEQ ID NO: 14, the CD8α hinge region nucleotide sequence is shown in SEQ ID NO: 15, the CD8 transmembrane region nucleotide sequence is shown in SEQ ID NO: 16, and the TLR7, CD40, and CD3ζ nucleotide sequences are shown in SEQ ID NO: 19, SEQ ID NO: 23, and SEQ ID NO:

26.

2. The CAR-Ms according to claim 1, wherein The single-chain variable fragment is derived from a single-chain antibody of MUC1, and includes a light chain variable region and a heavy chain variable region, which are connected by a linker sequence.

3. Use of the CAR-Ms according to claim 1 or 2 in the preparation of an anti-tumor pharmaceutical composition.

4. The use of CAR-Ms in preparing an anti-tumor pharmaceutical composition according to claim 3, characterized in that: The tumor is selected from at least one of lung cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, nervous system cancer, leukemia, and cervical cancer.

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